The long-term goals of the University of Pennsylvania Nanomedicine Development Center (NDC) are to characterize quantitatively at the molecular scale the components and functional organization of selected supra-molecular cellular compartments (SMCCs), and to develop the nanoscale tools required to repair or replace these components in diseased cells. Many developmental and acquired diseases are caused by deficits in assembly or repair of specialized SMCCs. The molecular details of how cells accomplish the programmed self-assembly of complex compartments, such as photoreceptor outer segments, primary cilia in the renal tubules, the nerve growth cone, and the myofibril, have tremendous health-care relevance and intrinsic nanotechnological interest. We will combine state-of-the-art molecular biologic techniques with both existing and new nanotechniques to investigate the specific requirements of the individual SMCC components, the order of their assembly, their intramolecular contacts and interactions, and the crucial involvement of accessory proteins, such as chaperones and molecular motors. Improved understanding of these details will have two important outcomes: Firstly, replacement of defective components will be more readily targeted in disease processes, such as retinal degenerations, polycystic kidney disease and cardiomyopathy. Secondly, full understanding of the assembly requirements will allow design of practical, artificial nanotechnological devices, such as sensors, actuators and delivery vehicles, using the same principles of specificity and self-assembly. We will refine and develop several nanomedical tools for the proposed research. For example, we will perfect the two-photon confocal microscopy techniques required to quantitate individual SMCC components and follow their movements in real time within living cells. We will develop the use of synthetic polymersome nanoparticles to deliver fluorescently labeled SMCC proteins to living cells on the microscope stage for these experiments. We will use total internal reflection epifluorescence microscopy (TIRF) techniques to monitor cell-free assembly of SMCC component nanomachines. The use of modified polymersome and viral vector nanoparticles will be developed for delivery of essential SMCC components to cells in culture and in vivo. In addition, we will investigate new techniques for measurement of intracellular assembly of SMCC components at high resolution using nanotubes as optical sensors. These research tools will be useful for the study of multiple SMCCs, and broadly applicable to nanomedicine research in general.